P2-Receptors (P2-Rs) represent significant targets for novel drug development. P2-Rs were identified also on pancreatic B cells and are involved in insulin secretion. Therefore, novel P2Y-R ligands, 2-thioether 5'-O-phosphorothioate adenosine derivatives (2-RS-ATP-alpha-S), were synthesized as potential insulin secretagogues. An efficient synthesis of these nucleotides and a facile method for separation of the chiral products are described. The enzymatic stability of the compounds toward pig pancreas type I ATPDase was evaluated. The rate of hydrolysis of 2-hexylthio-5'-O-(1-thiotriphosphate)adenosine (2-hexylthio-ATP-alpha-S) isomers by ATPDase was 28% of that of ATP. Some 2-thioether 5'-(monophosphorothioate)adenosine derivatives (2-RS-AMP-S) exerted an inhibitory effect on ATPDase. The apparent affinity of the compounds to P2Y(1)-R was determined by measurement of P2Y-R-promoted phospholipase C activity in turkey erythrocyte membranes. 2-RS-ATP-alpha-S derivatives were agonists, stimulating the production of inositol phosphates with K(0.5) values in the nanomolar range. 2-RS-AMP-S derivatives were full agonists, although 2 orders of magnitude less potent. All the compounds were more potent than ATP. The effect on insulin secretion and pancreatic flow rate was evaluated on isolated and perfused rat pancreas. A high increase, up to 500%, in glucose-induced insulin secretion was due to addition of 2-hexylthio-ATP-alpha-S in the nanomolar concentration range, which represents 100-fold enhancement of activity relative to ATP. 2-Hexylthio-AMP-S was 2.5 orders of magnitude less effective.